Spray dried microbes and methods of preparation and use

ABSTRACT

The invention provides spray-dried preparations of microbes and methods of using those microbes.

RELATED APPLICATION

This patent application is a U.S. National Stage Filing under 35 U.S.C.371 from International Patent Application Serial No. PCT/US2007/024074,filed Nov. 16, 2007, and published on May 22, 2009, as WO 2009/064277A1, the contents of which are incorporated herein by reference in theirentirety.

BACKGROUND

Glyoxylic acid and other keto acids such as pyruvic acid are importantintermediates in the manufacture of various agrochemicals,pharmaceuticals and fragrances. Typical commercial production ofglyoxylic acid employs either oxidation chemistry or electrochemistry.Electrochemical manufacture involves either the reduction of oxalic acidor the anodic oxidation of glyoxal to form glyoxylic acid whereaschemical oxidation generally involves the oxidation of glyoxal in thepresence of a strong acid such as HNO₃. A consequence of thesecommercial processes is the production of waste streams containingvarious toxic acids and heavy metals.

Glycolate oxidase is an enzyme available from various sources, includinggreen leafy plants and mammalian cells, which catalyzes the oxidation ofglycolic acid to glyoxylic acid, with the concomitant production ofhydrogen peroxide. For instance, Tolbert et al., J. Biol. Chem., 181:905(1949), reported an enzyme, extracted from tobacco leaves, whichcatalyzed the oxidation of glycolic acid to formic acid and CO₂ via theintermediate formation of glyoxylic acid. The addition of certaincompounds, such as ethylenediamine, limited the further oxidation of theintermediate glyoxylic acid. The oxidations were carried out at a pH ofabout 8, typically using glycolic acid concentrations of about 3-40 mM(millimolar). The optimum pH for the glycolate oxidation was reported tobe pH 8.9. Oxalic acid (100 mM) was reported to inhibit the catalyticaction of the glycolate oxidase. Similarly, Richardson and Tolbert, J.Biol. Chem., 236:1280 (1961), reported the formation of oxalic acidduring the glycolate oxidase-catalyzed oxidation of glycolic acid toglyoxylic acid, using enzymes isolated from tobacco, sugar beet, Swisschard, spinach, or rat liver. Richardson and Tolbert, J. Biol. Chem.,236:1280 (1961), also showed that buffers containingtris(hydroxymethyl)aminomethane (TRIS) inhibited the formation of oxalicacid in the glycolate oxidase catalyzed oxidation of glycolic acid.Clagett et al., J. Biol. Chem., 78:977 (1949) reported that the optimumpH for the glycolate oxidase catalyzed oxidation of glycolic acid withoxygen was about 7.8-8.6, and the optimum temperature was 35°-40° C.

Recent advances in recombinant DNA technology, combined with theisolation of cDNA coding for the spinach glycolate oxidase (see Volokitaet al, J. Biol. Chem., 262:15825 (1987)), have allowed for theconstruction of microbial strains that are intended to serve asalternative, economic enzyme sources. For instance, yeast offers severaladvantages to commercial applications over Escherichia coli and otherbacteria. Yeast can generally be grown to higher densities than bacteriaand are readily adaptable to continuous fermentation processing. It hasbeen reported, for example, that Pichia pastoris can be grown to celldensities in excess of 100 g/L (U.S. Pat. No. 4,414,329). Additionaladvantages of yeast hosts include the fact that many critical functionsof the organism, such as oxidative phosphorylations, are located withinorganelles and thus are not exposed to the possible deleterious effectsof the overexpression of foreign enzymatic products. Furthermore, yeastsappear to be capable of glycosylation of expressed polypeptide products,where such glycosylation is important to the bioactivity of thepolypeptide product.

Zelitch and Ochoa, J. Biol. Chem., 201:707 (1953), and Robinson et al.,J. Biol. Chem., 237:2001 (1962), reported that the formation of formicacid and CO₂ in the spinach glycolate oxidase-catalyzed oxidation ofglycolic acid resulted from the nonenzymatic reaction of H₂O₂ withglyoxylic acid. They observed that addition of catalase, an enzyme thatcatalyzes the decomposition of H₂O₂, greatly improved the yields ofglyoxylic acid by suppressing the formation of formic acid and CO₂. Withrespect to glyoxylic acid production in yeast, glyoxylic acid wasproduced when glycolic acid and oxygen were reacted in an aqueoussolution in the presence of aminomethylphosphonic acid and a catalystwhich is a genetically-engineered microbial yeast transformant whichexpresses the enzyme glycolate oxidase from spinach ((S)-2-hydroxy-acidoxidase, EC 1.1.3.15), and catalase (EC 1.11.1.6) (see U.S. Pat. No.5,693,490).

SUMMARY OF THE INVENTION

The invention provides spray-dried preparations of microbial cells suchas bacterial and yeast cells, or lysates or crude extracts thereof,suitable for biocatalysis. Spray-drying of microbial cells, such asPichia, Saccharomyces or E. coli, renders the cells suitable forbiocatalysis via opening of the cell wall or cell membrane in theabsence of permeabilization agents allowing for substrates and productsto freely move in and out of the cells. Thus, one or more enzymes usefulin biocatalysis may be expressed in microbes, e.g., recombinantlyexpressed, and those cells spray-dried. Spray drying results inmicrobial cells that are porous but have structural integrity, e.g.,enzymes do not readily leach out of spray-dried cells, in enzymes thatare stable at room temperature, and permits processing in water ratherthan a buffer at a particular pH. The stability and lack of leaching ofenzymes from spray-dried microbial cells allows for repeated use of thecells over time, e.g., continuous and extended use of a singlepreparation of spray-dried cells. In one embodiment, spray-driedmicrobial cells having at least two enzymes for a coupled reaction areprovided. In one embodiment, spray-dried microbial cells, e.g., yeastcells suitable for pyruvate or glycolate production, such as thoseexpressing glycolate oxidase and catalase, are provided. In oneembodiment, spray-dried microbial cells such as yeast cells suitable forpyruvate or glycolate production have recombinant glycolate oxidase,e.g., a heterologous glycolate oxidase. In one embodiment, spray-driedmicrobial cells suitable for pyruvate or glycolate production haverecombinant catalase, e.g., a heterologous catalase. For example, in oneembodiment, spray-dried Pichia cells have spinach glycolate oxidase andSacharromyces catalase T. However, the invention is not limited toparticular microbes and/or particular enzyme catalyzed reactions, as itis contemplated that microbes generally, whether recombinant ornonrecombinant, may be spray-dried and result in a source of stableenzyme(s) for biocatalysis.

The use of spray-dried microbial cells may reduce the number of processsteps for biocatalysis-based product production. For instance, thenumber of steps for glycolate oxidase-mediated production of severalproducts has been reduced by at least 2- to 3-fold using spray-driedmicrobial cells, thereby providing a simpler process and significantcost advantages. In one embodiment, a microbial cell suspension may bespray-dried directly from the fermentor, thus eliminating a solid/liquidseparation step. For example, the cell containing solution directly fromthe fermentor (without separation) may be spray-dried or first subjectedto cell separation and resuspension, e.g., in water or a buffer, priorto spray drying.

The invention provides a method to prepare a whole microbial cellbiocatalyst preparation. The method includes providing a microbial cellsuspension and spray drying the microbial cell suspension underconditions effective to yield a spray-dried microbial cell preparationsuitable for biocatalysis. In one embodiment, a spray-dried preparationof yeast is provided. In one embodiment, a spray-dried preparation ofPichia or Saccharomyces is provided In one embodiment, the spray-driedpreparation of yeast has at least one recombinant enzyme encoded by anexpression cassette having a promoter operably linked to an open readingframe for the enzyme. The methods for preparing the spray-driedmicrobial cell biocatalyst surprisingly do not require detergent orother membrane permeabilization agent treatment, e.g., do not require acationic surfactant, such as benzylalkonium chloride (BAC), treatment,or enzyme stabilizing agents such as sucrose, trehalose, thiols or otherpolyols.

Further provided is a method to prepare products of an enzymaticreaction. The method includes combining a spray-dried preparation of theinvention and an aqueous solution comprising a substrate for an enzymepresent in the spray-dried preparation under conditions that yield oneor more products of the enzyme catalyzed reaction. In one embodiment,the product is pyruvic acid or its salts, which are useful in food,pharmaceutical and agrochemical products, e.g., in dietary supplements,cosmetics or oxygenated solvents, and production of amino acids,hydrazone, indoxocarb and indole-3-pyruvic acid.

For example, a spray-dried preparation of yeast having recombinantglycolate oxidase is combined with an aqueous solution comprising asubstrate for the enzyme under conditions that yield a product of theenzyme catalyzed reaction. Products of the reaction, for instance,pyruvate, glyoxylate, keto acids or chiral hydroxyl acids, are thenisolated from the aqueous medium.

In one embodiment, the invention provides spray-dried preparations ofmicrobes having enzymes that provide chiral resolution or enantiomericselectivity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Glycolate oxidase reactions.

FIG. 2A. Process for producing products of the glycolate oxidasereaction using yeast and benzylalkonium chloride (BAC) treatment.

FIG. 2B. Process for producing products of the glycolate oxidasereaction using spray-dried yeast cells.

FIG. 3. Glycolate oxidase activity versus temperature.

FIG. 4. Glycolate oxidase activity versus feed rate.

FIG. 5. Glycolate oxidase activity versus feed concentration.

FIG. 6. Catalase activity versus temperature.

FIG. 7. Catalase activity versus feed rate.

FIG. 8. Catalase activity versus feed concentration.

FIG. 9. Glycolate oxidase activity versus temperature.

FIG. 10. Glycolate oxidase activity versus feed concentration.

FIG. 11. Glycolate oxidase activity versus temperature*feedconcentration.

FIG. 12. Catalase activity versus temperature.

FIG. 13. Catalase activity versus feed concentration.

FIG. 14. Catalase activity versus temperature*feed concentration.

FIG. 15. Glycolate oxidase activity in spray-dried yeast cells.

FIG. 16. Catalase activity in spray-dried yeast cells.

FIG. 17. Glycolate oxidase stability in spray-dried yeast cells.

FIG. 18. Catalase stability in spray-dried yeast cells.

FIG. 19. Glycolate oxidase activity in spray-dried yeast cells that havebeen detergent permeabilized.

FIG. 20. Catalase activity in spray-dried yeast cells that have beendetergent permeabilized.

FIG. 21. Testing for the presence or absence of glycolate oxidaseleaching from spray-dried yeast cells.

FIG. 22. Testing for the presence or absence of catalase leaching fromspray-dried yeast cells.

FIG. 23. Enantiomeric specificity of spray-dried yeast cells withglycolate oxidase and catalase.

FIG. 24. Analysis of E. coli lysates. Lane 1) 0.5 μL insoluble fraction*of Rosetta gami B(DE3)/pET-GO#11; lane 2) MW marker; lane 3) 2.0 μL cellextract of Rosetta gami B(DE3)/pET-GO#11 (7.4 μg of protein); lane 4)2.0 μL insoluble fraction of Rosetta gami B(DE3)/pET-GO#11; lane 5) 5.3μL cell extract of Rosetta gami B(DE3)/pET-32a, a control strain (7.4 μgof protein); and lane 6) 5.3 μL insoluble fraction of Rosetta gamiB(DE3)/pET-32a. * After lysing the cells by French Press, the lysate wascentrifuged at 25,800×g for 10 minutes, and the resulting supernatant(about 10 mL) was saved as the cell extract. The pellet was suspended in10 mL PBS and considered to be the insoluble fraction.

FIGS. 25A-B. A) Glycolate oxidase activity in Rosetta gamiB(DE3)/pET-GO#11 cell extract. B) Glycolate oxidase activity in Rosettagami B(DE3) cell extract.

FIG. 26. Glycolate oxidase activity in spray-dried or blotted Rosettagami B(DE3)/pET-GO#11 cells.

FIG. 27. Catalase activity in spray-dried or control S. cerevisiaecells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “nucleic acid molecule”, “polynucleotide”, or “nucleic acidsequence” as used herein, refers to nucleic acid, DNA or RNA, thatcomprises coding sequences necessary for the production of a polypeptideor protein precursor. The encoded polypeptide may be a full-lengthpolypeptide, a fragment thereof (less than full-length), or a fusion ofeither the full-length polypeptide or fragment thereof with anotherpolypeptide, yielding a fusion polypeptide.

A “nucleic acid”, as used herein, is a covalently linked sequence ofnucleotides in which the 3′ position of the pentose of one nucleotide isjoined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence, i.e., a linear order of nucleotides. A“polynucleotide”, as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. An“oligonucleotide” or “primer”, as used herein, is a short polynucleotideor a portion of a polynucleotide. An oligonucleotide typically containsa sequence of about two to about one hundred bases. The word “oligo” issometimes used in place of the word “oligonucleotide”.

Nucleic acid molecules are said to have a “5′-terminus” (5′ end) and a“3′-terminus” (3′ end) because nucleic acid phosphodiester linkagesoccur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a polynucleotide at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a polynucleotide at which a new linkage would be to a 3′ carbon isits 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring.

As used herein, a nucleic acid sequence, even if internal to a largeroligonucleotide or polynucleotide, also may be said to have 5′ and 3′ends. In either a linear or circular DNA molecule, discrete elements arereferred to as being “upstream” or 5′ of the “downstream” or 3′elements. This terminology reflects the fact that transcription proceedsin a 5′ to 3′ fashion along the DNA strand. Typically, promoter andenhancer elements that direct transcription of a linked gene (e.g., openreading frame or coding region) are generally located 5′ or upstream ofthe coding region. However, enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region.Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

The term “codon” as used herein, is a basic genetic coding unit,consisting of a sequence of three nucleotides that specify a particularamino acid to be incorporated into a polypeptide chain, or a start orstop signal. The term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. Typically, the coding region is bounded on the 5′ side bythe nucleotide triplet “ATG” which encodes the initiator methionine andon the 3′ side by a stop codon (e.g., TAA, TAG, TGA). In some cases thecoding region is also known to initiate by a nucleotide triplet “TTG”.

The term “gene” refers to a DNA sequence that comprises coding sequencesand optionally control sequences necessary for the production of apolypeptide from the DNA sequence.

As used herein, the term “heterologous” nucleic acid sequence or proteinrefers to a sequence that relative to a reference sequence has adifferent source, e.g., originates from a foreign species, or, if fromthe same species, it may be substantially modified from the originalform.

Nucleic acids are known to contain different types of mutations. A“point” mutation refers to an alteration in the sequence of a nucleotideat a single base position from the wild-type sequence. Mutations mayalso refer to insertion or deletion of one or more bases, so that thenucleic acid sequence differs from a reference, e.g., a wild-type,sequence.

As used herein, the terms “hybridize” and “hybridization” refer to theannealing of a complementary sequence to the target nucleic acid, i.e.,the ability of two polymers of nucleic acid (polynucleotides) containingcomplementary sequences to anneal through base pairing. The terms“annealed” and “hybridized” are used interchangeably throughout, and areintended to encompass any specific and reproducible interaction betweena complementary sequence and a target nucleic acid, including binding ofregions having only partial complementarity. Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Those skilled in the art of nucleic acid technology candetermine duplex stability empirically considering a number of variablesincluding, for example, the length of the complementary sequence, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs. The stability of a nucleic acidduplex is measured by the melting temperature, or “T_(m)”. The T_(m) ofa particular nucleic acid duplex under specified conditions is thetemperature at which on average half of the base pairs havedisassociated.

The term “vector” is used in reference to nucleic acid molecules intowhich fragments of DNA may be inserted or cloned and can be used totransfer DNA segment(s) into a cell and capable of replication in acell. Vectors may be derived from plasmids, bacteriophages, viruses,cosmids, and the like.

The terms “recombinant vector” and “expression vector” as used hereinrefer to DNA or RNA sequences containing a desired coding sequence andappropriate DNA or RNA sequences necessary for the expression of theoperably linked coding sequence in a particular host organism.Prokaryotic expression vectors include a promoter, a ribosome bindingsite, an origin of replication for autonomous replication in a host celland possibly other sequences, e.g. an optional operator sequence,optional restriction enzyme sites. A promoter is defined as a DNAsequence that directs RNA polymerase to bind to DNA and to initiate RNAsynthesis. Eukaryotic expression vectors include a promoter, optionallya polyadenylation signal and optionally an enhancer sequence.

A polynucleotide having a nucleotide sequence encoding a protein orpolypeptide means a nucleic acid sequence comprising the coding regionof a gene, or in other words the nucleic acid sequence encodes a geneproduct. The coding region may be present in either a cDNA, genomic DNAor RNA form. When present in a DNA form, the oligonucleotide may besingle-stranded (i.e., the sense strand) or double-stranded. Suitablecontrol elements such as enhancers/promoters, splice junctions,polyadenylation signals, etc. may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers/promoters, splicejunctions, intervening sequences, polyadenylation signals, etc. Infurther embodiments, the coding region may contain a combination of bothendogenous and exogenous control elements.

The term “transcription regulatory element” or “transcription regulatorysequence” refers to a genetic element or sequence that controls someaspect of the expression of nucleic acid sequence(s). For example, apromoter is a regulatory element that facilitates the initiation oftranscription of an operably linked coding region. Other regulatoryelements include, but are not limited to, transcription factor bindingsites, splicing signals, polyadenylation signals, termination signalsand enhancer elements.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription. Promoter and enhancer elements have been isolated froma variety of eukaryotic sources including genes in yeast, insect andmammalian cells. Promoter and enhancer elements have also been isolatedfrom viruses and analogous control elements, such as promoters, are alsofound in prokaryotes. The selection of a particular promoter andenhancer depends on the cell type used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types. Forexample, the SV40 early gene enhancer is very active in a wide varietyof cell types from many mammalian species and has been widely used forthe expression of proteins in mammalian cells. Two other examples ofpromoter/enhancer elements active in a broad range of mammalian celltypes are those from the human elongation factor 1 gene and the longterminal repeats of the Rous sarcoma virus; and the humancytomegalovirus.

The term “promoter/enhancer” denotes a segment of DNA containingsequences capable of providing both promoter and enhancer functions(i.e., the functions provided by a promoter element and an enhancerelement as described above). For example, the long terminal repeats ofretroviruses contain both promoter and enhancer functions. Theenhancer/promoter may be “endogenous” or “exogenous” or “heterologous.”An “endogenous” enhancer/promoter is one that is naturally linked with agiven gene in the genome. An “exogenous” or “heterologous”enhancer/promoter is one that is placed in juxtaposition to a gene bymeans of genetic manipulation (i.e., molecular biological techniques)such that transcription of the gene is directed by the linkedenhancer/promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site. A commonly used splice donor and acceptor site is thesplice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly(A) site” or“poly(A) sequence” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable, astranscripts lacking a poly(A) tail are unstable and are rapidlydegraded. The poly(A) signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly(A) signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly(A) signal is one which has been isolatedfrom one gene and positioned 3′ to another gene. A commonly usedheterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A)signal is contained on a 237 bp BamHI/Bcl I restriction fragment anddirects both termination and polyadenylation.

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors containingeither the SV40 or polyoma virus origin of replication replicate to highcopy number (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. In contrast, vectors containing thereplicons from bovine papillomavirus or Epstein-Barr virus replicateextrachromosomally at low copy number (about 100 copies/cell).

The term “in vitro” refers to an artificial environment and to processesor reactions that occur within an artificial environment. In vitroenvironments include, but are not limited to, test tubes and celllysates. The term “in vivo” refers to the natural environment (e.g., ananimal or a cell) and to processes or reaction that occur within anatural environment.

The term “expression system” refers to any assay or system fordetermining (e.g., detecting) the expression of a gene of interest.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used. A wide range ofsuitable mammalian cells are available from a wide range of source(e.g., the American Type Culture Collection, Rockland, Md.). The methodof transformation or transfection and the choice of expression vehiclewill depend on the host system selected. Transformation and transfectionmethods are well known to the art. Expression systems include in vitrogene expression assays where a gene of interest (e.g., a reporter gene)is linked to a regulatory sequence and the expression of the gene ismonitored following treatment with an agent that inhibits or inducesexpression of the gene. Detection of gene expression can be through anysuitable means including, but not limited to, detection of expressedmRNA or protein (e.g., a detectable product of a reporter gene) orthrough a detectable change in the phenotype of a cell expressing thegene of interest. Expression systems may also comprise assays where acleavage event or other nucleic acid or cellular change is detected.

The term “wild-type” as used herein, refers to a gene or gene productthat has the characteristics of that gene or gene product isolated froma naturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “wild-type” form of the gene. In contrast, the term “mutant” refersto a gene or gene product that displays modifications in sequence and/orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

The term “isolated” when used in relation to a nucleic acid, as in“isolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant with which it is ordinarily associated in its source. Thus,an isolated nucleic acid is present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids (e.g., DNA and RNA) are found in the statethey exist in nature. For example, a given DNA sequence (e.g., a gene)is found on the host cell chromosome in proximity to neighboring genes;RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAsthat encode a multitude of proteins. However, isolated nucleic acidincludes, by way of example, such nucleic acid in cells ordinarilyexpressing that nucleic acid where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid or oligonucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acidor oligonucleotide is to be utilized to express a protein, theoligonucleotide contains at a minimum, the sense or coding strand (i.e.,the oligonucleotide may single-stranded), but may contain both the senseand anti-sense strands (i.e., the oligonucleotide may bedouble-stranded).

By “peptide,” “protein” and “polypeptide” is meant any chain of aminoacids, regardless of length or post-translational modification (e.g.,glycosylation or phosphorylation). The nucleic acid molecules of theinvention may also encode a variant of a naturally-occurring protein orpolypeptide fragment thereof, which has an amino acid sequence that isat least 85%, 90%, 95% or 99% identical to the amino acid sequence ofthe naturally-occurring (native or wild-type) protein from which it isderived. The term “fusion polypeptide” or “fusion protein” refers to achimeric protein containing a reference protein (e.g., luciferase)joined at the N- and/or C-terminus to one or more heterologous sequences(e.g., a non-luciferase polypeptide). In some embodiments, a modifiedpolypeptide, fusion polypeptide or a portion of a full-lengthpolypeptide of the invention, may retain at least some of the activityof a corresponding full-length functional (nonchimeric) polypeptide. Inother embodiments, in the absence of an exogenous agent or molecule ofinterest, a modified polypeptide, fusion polypeptide or portion of afull-length functional polypeptide of the invention, may lack activityrelative to a corresponding full-length functional polypeptide. In otherembodiments, a modified polypeptide, fusion polypeptide or portion of afull-length functional polypeptide of the invention in the presence ofan exogenous agent may retain at least some or have substantially thesame activity, or alternatively lack activity, relative to acorresponding full-length functional polypeptide.

Polypeptide molecules are said to have an “amino terminus” (N-terminus)and a “carboxy terminus” (C-terminus) because peptide linkages occurbetween the backbone amino group of a first amino acid residue and thebackbone carboxyl group of a second amino acid residue. The terms“N-terminal” and “C-terminal” in reference to polypeptide sequencesrefer to regions of polypeptides including portions of the N-terminaland C-terminal regions of the polypeptide, respectively. A sequence thatincludes a portion of the N-terminal region of polypeptide includesamino acids predominantly from the N-terminal half of the polypeptidechain, but is not limited to such sequences. For example, an N-terminalsequence may include an interior portion of the polypeptide sequenceincluding bases from both the N-terminal and C-terminal halves of thepolypeptide. The same applies to C-terminal regions. N-terminal andC-terminal regions may, but need not, include the amino acid definingthe ultimate N-terminus and C-terminus of the polypeptide, respectively.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein.

The terms “cell,” “cell line,” “host cell,” as used herein, are usedinterchangeably, and all such designations include progeny or potentialprogeny of these designations. By “transformed cell” is meant a cellinto which (or into an ancestor of which) has been introduced a nucleicacid molecule of the invention. Optionally, a nucleic acid molecule ofthe invention may be introduced into a suitable cell line so as tocreate a stably-transfected cell line capable of producing the proteinor polypeptide encoded by the gene. Vectors, cells, and methods forconstructing such cell lines are well known in the art. The words“transformants” or “transformed cells” include the primary transformedcells derived from the originally transformed cell without regard to thenumber of transfers. All progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Nonetheless, mutantprogeny that have the same functionality as screened for in theoriginally transformed cell are included in the definition oftransformants.

The term “homology” refers to a degree of complementarity between two ormore sequences. There may be partial homology or complete homology(i.e., identity). Homology is often measured using sequence analysissoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group. University of Wisconsin Biotechnology Center. 1710University Avenue. Madison, Wis. 53705). Such software matches similarsequences by assigning degrees of homology to various substitutions,deletions, insertions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

The term “isolated” when used in relation to a polypeptide, as in“isolated protein” or “isolated polypeptide” refers to a polypeptidethat is identified and separated from at least one contaminant withwhich it is ordinarily associated in its source. Thus, an isolatedpolypeptide is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated polypeptides(e.g., proteins and enzymes) are found in the state they exist innature.

The term “purified” or “to purify” means the result of any process thatremoves some of a contaminant from the component of interest, such as aprotein or nucleic acid. The percent of a purified component is therebyincreased in the sample.

As used herein, “pure” means an object species is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a “substantially pure”composition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, about 90%, about 95%, and about 99%. Most preferably,the object species is purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of a singlemacromolecular species.

The term “operably linked” as used herein refer to the linkage ofnucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. The term alsorefers to the linkage of sequences encoding amino acids in such a mannerthat a functional (e.g., enzymatically active, capable of binding to abinding partner, capable of inhibiting, etc.) protein or polypeptide isproduced.

As used herein, a “marker gene” or “reporter gene” is a gene thatimparts a distinct phenotype to cells expressing the gene and thuspermits cells having the gene to be distinguished from cells that do nothave the gene. Such genes may encode either a selectable or screenablemarker, depending on whether the marker confers a trait which one can‘select’ for by chemical means, i.e., through the use of a selectiveagent (e.g., a herbicide, antibiotic, or the like), or whether it issimply a “reporter” trait that one can identify through observation ortesting, i.e., by ‘screening’. Elements of the present disclosure areexemplified in detail through the use of particular marker genes. Ofcourse, many examples of suitable marker genes or reporter genes areknown to the art and can be employed in the practice of the invention.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the alteration of any gene.Exemplary modified reporter proteins are encoded by nucleic acidmolecules comprising modified reporter genes including, but are notlimited to, modifications of a neo gene, a β-gal gene, a gus gene, a catgene, a gpt gene, a hyg gene, a hisD gene, a ble gene, a mprt gene, abar gene, a nitrilase gene, a galactopyranoside gene, a xylosidase gene,a thymidine kinase gene, an arabinosidase gene, a mutant acetolactatesynthase gene (ALS) or acetoacid synthase gene (AAS), amethotrexate-resistant dhfr gene, a dalapon dehalogenase gene, a mutatedanthranilate synthase gene that confers resistance to 5-methyltryptophan (WO 97/26366), an R-locus gene, a β-lactamase gene, a xylEgene, an α-amylase gene, a tyrosinase gene, a luciferase (luc) gene,(e.g., a Renilla reniformis luciferase gene, a firefly luciferase gene,or a click beetle luciferase (Pyrophorus plagiophthalamus) gene), anaequorin gene, a red fluorescent protein gene, or a green fluorescentprotein gene.

All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature,abbreviations for amino acid residues are as shown in the followingTable of Correspondence.

TABLE OF CORRESPONDENCE 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine GGly L-glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine SSer L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteineSources of Cells for Spray-Drying and Methods of Preparation and Use

The invention provides spray-dried preparations of microbial cells, orlysates or crude extracts thereof, suitable for biocatalysis, and asimpler process for using those cells, lysates or crude extractsthereof, in biocatalysis. In one embodiment of the invention, theinvention provides for spray-dried preparations of prokaryotic cells, orlysates or crude extracts thereof, suitable for biocatalysis, and asimpler process for using those cells, lysates or crude extractsthereof, in biocatalysis. In one embodiment, the prokaryotic cells areE. coli cells. In another embodiment, the prokaryotic cells arePseudomonas cells.

The invention also provides spray-dried preparations of yeast suitablefor biocatalysis and a simpler process for using those cells inbiocatalysis. For example, previously for production of pyruvate andglyoxylate by a recombinant P. pastoris having glycolate oxidase andcatalase, the cells were harvested, washed, treated with benzylalkoniumchloride (BAC) to make the cells porous to substrates such as lactateand glycolate, and washed several times to remove any residual BAC, andused for the production of pyruvate and glyoxylate (about 6 to 8 steps).

The present invention provides for a process to spray-dry microbialcells to render them porous and suitable for biocatalysis, withoutleaching of enzymes for the biocatalysis. Thus, the cells can be useddirectly for production. Accordingly, the process to take a biocatalystfrom the fermentor to the reactor has been simplified by several steps.Spray-drying the cells may also render the enzymes in those cellsstable. As described herein for recombinant cells with glycolate oxidaseand catalase, spray drying did not result in enzyme leaching or a needto treat with BAC. Also, spray drying resulted in stable enzymes and didnot compromise the selectivity of glycolate oxidase towards variousα-hydroxyacids, as the spray-dried enzyme is active on S-acids (i.e.,oxidizes S-acids) but is not active towards R-acids, i.e., thespray-dried enzyme is selective for S-acids not R-acids. Thus, kineticresolution of chiral compound, for example, racemic α-hydroxyacids isalso possible with the spray-dried preparations described herein.Accordingly, the invention provides microbial cells such as yeast cells,e.g., Pichia or Saccharomyces cells, as well as recombinant microbialcells, such as recombinant Pichia, Pseudomonas or E. coli cells, for theproduction of various chemicals and resolution of enantiomers. Thespray-dried cells are easy to prepare, store and use.

Yeast cells useful in the present invention are those from phylumAscomycota, subphylum Saccharomycotina, class Saccharomycetes, orderSaccharomycetales or Schizosaccharomycetales, family Saccharomycetaceae,genus Saccharomyces or Pichia (Hansenula), e.g., species: P. anomola, P.guilliermondiii, P. norvegenesis, P. ohmeri, and P. pastoris. Yeastcells employed in the invention may be native (non-recombinant) cells orrecombinant cells, e.g., those which are transformed with exogenous(recombinant) DNA having one or more expression cassettes each with apolynucleotide having a promoter and an open reading frame encoding oneor more enzymes useful for biocatalysis. The enzyme(s) encoded by theexogenous DNA is referred to as “recombinant,” and that enzyme may befrom the same species or heterologous (from a different species). Forexample, a recombinant P. pastoris cell may recombinantly express a P.pastoris enzyme or a plant, microbial, e.g., Aspergillus orSaccharomyces, or mammalian enzyme.

In one embodiment, the microbial cell employed in the methods of theinvention is transformed with recombinant DNA, e.g., in a vector.Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) BACs(bacterial artificial chromosomes) and DNA segments for use intransforming cells will generally comprise DNA encoding an enzyme, aswell as other DNA that one desires to introduce into the cells. TheseDNA constructs can further include elements such as promoters,enhancers, polylinkers, marker or selectable genes, or even regulatorygenes, as desired. For instance, one of the DNA segments or genes chosenfor cellular introduction will often encode a protein that will beexpressed in the resultant transformed (recombinant) cells, such as toresult in a screenable or selectable trait and/or that will impart animproved phenotype to the transformed cell. However, this may not alwaysbe the case, and the present invention also encompasses transformedcells incorporating non-expressed transgenes.

DNA useful for introduction into cells includes that which has beenderived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into cells. An example of DNA “derived” from asource, would be a DNA sequence that is identified as a useful fragmentwithin a given organism, and that is then chemically synthesized inessentially pure form. An example of such DNA “isolated” from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by biochemical means, e.g., enzymatically, such as by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering. Such DNA is commonly also referred to as “recombinant DNA.”

Therefore, useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived fromintroduced RNA. The introduced DNA may be or may not be a DNA originallyresident in the host cell genotype that is the recipient of the DNA(native or heterologous). It is within the scope of the invention toisolate a gene from a given genotype, and to subsequently introducemultiple copies of the gene into the same genotype, e.g., to enhanceproduction of a given gene product.

The introduced DNA includes, but is not limited to, DNA from genes suchas those from bacteria, yeasts, fungi, plants or vertebrates, e.g.,mammals. The introduced DNA can include modified or synthetic genes,e.g., “evolved” genes, portions of genes, or chimeric genes, includinggenes from the same or different genotype. The term “chimeric gene” or“chimeric DNA” is defined as a gene or DNA sequence or segmentcomprising at least two DNA sequences or segments from species that donot combine DNA under natural conditions, or which DNA sequences orsegments are positioned or linked in a manner that does not normallyoccur in the native genome of the untransformed cell.

The introduced DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, which can also contain codingregions flanked by regulatory sequences that promote the expression ofthe recombinant DNA present in the transformed cell. For example, theDNA may include a promoter that is active in a cell that is derived froma source other than that cell, or may utilize a promoter already presentin the cell that is the transformation target.

Generally, the introduced DNA will be relatively small, i.e., less thanabout 30 kb to minimize any susceptibility to physical, chemical, orenzymatic degradation that is known to increase as the size of the DNAincreases. The number of proteins, RNA transcripts or mixtures thereofthat is introduced into the cell is preferably preselected and defined,e.g., from one to about 5-10 such products of the introduced DNA may beformed.

The selection of an appropriate expression vector will depend upon thehost cells. An expression vector can contain, for example, (1)prokaryotic DNA elements coding for a bacterial origin of replicationand an antibiotic resistance gene to provide for the amplification andselection of the expression vector in a bacterial host; (2) DNA elementsthat control initiation of transcription such as a promoter; (3) DNAelements that control the processing of transcripts such as introns,transcription termination/polyadenylation sequence; and (4) a gene ofinterest that is operatively linked to the DNA elements to controltranscription initiation. The expression vector used may be one capableof autonomously replicating in the host cell or capable of integratinginto the chromosome, originally containing a promoter at a site enablingtranscription of the linked gene.

Yeast or fungal expression vectors may comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. Several well-characterized yeast expressionsystems are known in the art and described in, e.g., U.S. Pat. No.4,446,235, and European Patent Applications 103,409 and 100,561. A largevariety of shuttle vectors with yeast promoters are also known to theart. However, any other plasmid or vector may be used as long as theyare replicable and viable in the host.

The construction of vectors that may be employed in conjunction with thepresent invention will be known to those of skill of the art in light ofthe present disclosure (see, e.g., Sambrook and Russell, MolecularBiology: A Laboratory Manual, 2001). The expression cassette of theinvention may contain one or a plurality of restriction sites allowingfor placement of the polynucleotide encoding an enzyme. The expressioncassette may also contain a termination signal operably linked to thepolynucleotide as well as regulatory sequences required for propertranslation of the polynucleotide. The expression cassette containingthe polynucleotide of the invention may be chimeric, meaning that atleast one of its components is heterologous with respect to at least oneof the other components. Expression of the polynucleotide in theexpression cassette may be under the control of a constitutive promoter,inducible promoter, regulated promoter, viral promoter or syntheticpromoter.

The expression cassette may include, in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region,the polynucleotide of the invention and a transcriptional andtranslational termination region functional in vivo and/or in vitro. Thetermination region may be native with the transcriptional initiationregion, may be native with the polynucleotide, or may be derived fromanother source. The regulatory sequences may be located upstream (5′non-coding sequences), within (intron), or downstream (3′ non-codingsequences) of a coding sequence, and influence the transcription, RNAprocessing or stability, and/or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,enhancers, promoters, repressor binding sites, translation leadersequences, introns, and polyadenylation signal sequences. They mayinclude natural and synthetic sequences as well as sequences that may bea combination of synthetic and natural sequences.

The vector used in the present invention may also include appropriatesequences for amplifying expression.

A promoter is a nucleotide sequence that controls the expression of acoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. A promoter includes aminimal promoter, consisting only of all basal elements needed fortranscription initiation, such as a TATA-box and/or initiator that is ashort DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. A promoter maybe derived entirely from a native gene, or be composed of differentelements derived from different promoters found in nature, or even becomprised of synthetic DNA segments. A promoter may also contain DNAsequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions. A promoter may also include aminimal promoter plus a regulatory element or elements capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence contains of proximal and more distal elements,the latter elements are often referred to as enhancers.

Representative examples of promoters include, but are not limited to,promoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses. For instance, any promoter capable ofexpressing in yeast hosts can be used as a promoter in the presentinvention, for example, the GAL4 promoter may be used. Additionalpromoters useful for expression in a yeast cell are well described inthe art. Examples thereof include promoters of the genes coding forglycolytic enzymes, such as TDH3, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), a shortened version of GAPDH (GAPFL),3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase,phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglyceratemutase, pyruvate kinase, triosephosphate isomerase, phosphoglucoseisomerase, invertase and glucokinase genes and the like in theglycolytic pathway, heat shock protein promoter, MFa-1 promoter, CUP 1promoter, MET, the promoter of the TRP1 gene, the AOX (alcohol oxidase)gene promoter, e.g., the AOX1 or AOX2 promoter, the ADC1 gene (codingfor the alcohol dehydrogenase I) or ADR2 gene (coding for the alcoholdehydrogenase II), acid phosphatase (PHO5) gene, isocytochrome c gene, apromoter of the yeast mating pheromone genes coding for the a- orα-factor, or the GAL/CYC1 hybrid promoter (intergenic region of theGAL1-GAL10 gene/Cytochrome1 gene) (Guarente et al. 1982). Promoters withtranscriptional control that can be turned on or off by variation of thegrowth conditions include, e.g., PHO5, ADR2, and GAL/CYC1 promoters. ThePHO5 promoter, for example, can be repressed or derepressed at will,solely by increasing or decreasing the concentration of inorganicphosphate in the medium. Some promoters, such as the ADH1 promoter,allow high-level constitutive expression of the gene of interest.

Any promoter capable of expressing in filamentous fungi may be used.Examples are a promoter induced strongly by starch or cellulose, e.g., apromoter for glucoamylase or a-amylase from the genus Aspergillus orcellulase (cellobiohydrase) from the genus Trichoderma, a promoter forenzymes in the glycolytic pathway, such as phosphoglycerate kinase (pgk)and glycerylaldehyde 3-phosphate dehydrogenase (gpd), etc.

Particular bacterial promoters include but are not limited to E. colilac or trp, the phage lambda P_(L), lacZ, T3, T7, gpt, and lambda P_(R)promoters.

Two principal methods for the control of expression are known, viz.:induction, which leads to overexpression, and repression, which leads tounderexpression. Overexpression can be achieved by insertion of a strongpromoter in a position that is operably linked to the target gene, or byinsertion of one or more than one extra copy of the selected gene. Forexample, extra copies of the gene of interest may be positioned on anautonomously replicating plasmid, such as pYES2.0 (Invitrogen Corp.,Carlsbad, Calif.), where overexpression is controlled by the GAL4promoter after addition of galactose to the medium.

Several inducible promoters are known in the art. Many are described ina review by Gatz, Curr. Op. Biotech., 7:168 (1996) (see also Gatz, Ann.Rev. Plant. Physiol. Plant Mol. Biol., 48:89 (1997)). Examples includetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1a system),glucocorticoid-inducible (Aoyama T. et al., 1997), alcohol-induciblesystems, e.g., AOX promoters, and ecdysome-inducible systems. Alsoincluded are the benzene sulphonamide-inducible (U.S. Pat. No.5,364,780) and alcohol-inducible (WO 97/06269 and WO 97/06268) induciblesystems and glutathione S-transferase promoters.

In addition to the use of a particular promoter, other types of elementscan influence expression of transgenes. In particular, introns havedemonstrated the potential for enhancing transgene expression.

Other elements include those that can be regulated by endogenous orexogenous agents, e.g., by zinc finger proteins, including naturallyoccurring zinc finger proteins or chimeric zinc finger proteins. See,e.g., U.S. Pat. No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311.

An enhancer is a DNA sequence that can stimulate promoter activity andmay be an innate element of the promoter or a heterologous elementinserted to enhance the level or tissue specificity of a particularpromoter. An enhancer is capable of operating in both orientations (5′to 3′ and 3′ to 5′ relative to the gene of interest coding sequences),and is capable of functioning even when moved either upstream ordownstream from the promoter. Both enhancers and other upstream promoterelements bind sequence-specific DNA-binding proteins that mediate theireffects.

Vectors for use in accordance with the present invention may beconstructed to include an enhancer element. Constructs of the inventionwill also include the gene of interest along with a 3′ end DNA sequencethat acts as a signal to terminate transcription and allow for thepolyadenylation of the resultant mRNA.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose that include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence that may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure.

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait thatone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., an antibiotic, or the like), or whether it issimply a trait that one can identify through observation or testing,i.e., by ‘screening’. Of course, many examples of suitable marker genesare known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes are alsogenes that encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers that encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes that can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., by ELISAand small active enzymes detectable in extracellular solution.

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) that encode an enzyme for whichvarious chromogenic substrates are known; a beta-lactamase gene(Sutcliffe, 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky et al., 1983), which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an alpha-amylase gene (Ikuta et al.,1990); a tyrosinase gene (Katz et al., 1983) that encodes an enzymecapable of oxidizing tyrosine to DOPA and dopaquinone that in turncondenses to form the easily detectable compound melanin; abeta-galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), whichallows for bioluminescence detection; or even an aequorin gene (Prasheret al., 1985), which may be employed in calcium-sensitivebioluminescence detection, or a green fluorescent protein gene (Niedz etal., 1995). Selectable nutritional markers may also be used, such asHIS3, URA3, TRP-1, LYS-2 and ADE2.

Any construct encoding a gene product that results in a recombinant celluseful in biocatalysis may be employed. In one embodiment, the constructencodes an enzyme. Sources of genes for enzymes include those fromfungal cells belonging to the genera Aspergillus, Rhizopus, Trichoderma,Neurospora, Mucor, Penicillium, yeast belonging to the generaKluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon,Schwanniomyces, plants, vertebrates and the like. In one embodiment, theconstruct is on a plasmid suitable for extrachromosomal replication andmaintenance. In another embodiment, two constructs are concurrently orsequentially introduced to a cell so as to result in stable integrationof the constructs into the genome.

Enzymes useful in biocatalysis include but are not limited to all sixclasses of enzymes including hydrolases, lyases, isomerases, ligases,oxidoreductases and transferases, e.g., amidases, alcohol oxidases,carboxylases, dioxygenases, dismutases, demethylases, dehalogenases,dehydrogenases, deaminases, decarboxylases, desulfurases, epoxidases,hydrogenases, hydroxylases, hydratases, kinases, laccases, lactamases,mutases, nitrilases, nitrogenases, oxidases, peroxidases, phosphatases,reductases, racemases, synthetases, thiol oxidases, thiolases, andureases, and including but not limited to an amylase, aminopeptidase,amyloglucosidase, bromelain, carboxypeptidase, catalase, cellulase,collagenase, esterase, ferulic acid esterase, galactosidase, glucanase,glucoamylase, glucose oxidase, glucosidase, glycolate oxidase,hemicellulase, invertase, lactase, lipase, maltase, papain, pancreatin,pancreatic lipase, pectinase, pectin lyase, pentosanase, pepsin,peptidase, phospholipase A2, polygalacturonase, proteinase, sucrase,trypsin, xylanase, and xylanase. For instance, transketolase andtransaminase may be used to prepare chiral amino alcohols, ketones maybe converted to secondary alcohols by alcohol dehydrogenase,dentoxifyiline may be converted to lisofylline by alcohol dehydrogenase,and 3-alkyl cyclic ketones may be converted to 4- and 5-substitutedlactones by cyclopentanene oxygenase.

In one embodiment, the microbial cells of the invention express4-hydroxyphenylacetate 3-hydroxylase and in the presence of L-tyrosine,NADH and oxygen, produce L-dihydroxyphenylalanine. In one embodiment,the microbial cells of the invention express glucose, alcohol or formatedehydrogenase and an oxygenase that requires NAD(P) or NAD(P)H. Forexample, cells that express formate dehydrogenase may be combined withCO₂ to produce formic acid; cells that express glucose dehydrogenase maybe combined with gluconolactone to produce glucose; cells that expressalcohol dehydrogenase may be combined with acetalaldehyde to produceethanol; and cells that express phosphite dehydrogenase may be combinedwith phosphite to produce phosphate. In one embodiment, the biocatalyticreaction includes two different enzymes (a “coupled” reaction). In oneembodiment, cells that express nitrile hydratase and penicillin Gacylase may be employed to produce cephalexin; cells that expressalcohol dehydrogenase and NAD⁺-dependent formate dehydrogenase may beused to produce methyl (R)-3-hydroxybutanoate from methyl acetoacetate;cells that express alcohol or formate dehydrogenase and a monooxygenasemay be employed for regio- and stereo-selective biotransformations oflactones (see, e.g., Grogan et al., Biotech. Lett., 14:1125 (1992)). Inone embodiment, the cells of the invention express NAD(P)H-flavinoxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase, which areuseful in antibiotic biosynthesis.

In one embodiment, microbial cells such as yeast cells, e.g., Pichiacells, are spray-dried and employed for production of pyruvate,glyoxylate, resolution of racemic hydroxyacids, and/or production ofother keto-acids that glycolate oxidase is capable of oxidizing. In oneembodiment, the microbial cells express recombinant glycolate oxidase.Glycolate oxidase includes hydroxy-acid oxidase A and hydroxy-acidoxidase B (L-α-hydroxy acid oxidase, L-2-hydroxy acid oxidase,(S)-2-hydroxy-acid; oxygen 2-oxidoreductase). Glycolate oxidase is aflavoprotein (FMN), and the A form preferentially oxidizes short-chainaliphatic hydroxyl acids, while the B form preferentially oxidizeslong-chain and aromatic hydroxyl acids. Glycolate oxidases within thescope of the invention include but are not limited to those from plants,e.g., including but not limited to those from Solanum, Arabidopsis,Medicago, Fragiara, Spinacia, and Hordeum, or other sources, e.g., fromE. coli, Brucella, Ralsoria, Psuedomonus, Frankia, Bradyrhizobium,Xanthomonas, Synechococcus, Thermosynechochoccus, Gloeobacter, Bacillus,Agrobacterium, Mesorhizobium, Synechocytis, Aspergillus, or vertebratesincluding mammals such as bovine, rat, human, mouse and rat.

In one embodiment, the microbial cells, e.g., yeast cells, expressrecombinant catalase, optionally in addition to recombinant glycolateoxidase. Catalases may exhibit both catalase and peroxidase activity.Catalases within the scope of the invention include but are not limitedto catalases from Saccharomyces, Debaromyces, Yarrowsia, orKluyvermyces, and those disclosed in NCBI Accession Nos. P83657,CAT1_COMTR; Q9C168, CAT1_NEUCR; P81138, CAT1_PENJA; Q8X182, CAT2_NEUCR;Q9C169, CAT3_NEUCR; Q96528, CATA1_ARATH; Q27487, CATA1_CAEEL; P48350,CATA1_CUCPE; P17598, CATA1_GOSHI; P55307, CATA1_HORVU; P18122,CATA1_MAIZE; P49315, CATA1_NICPL; P00549, CATA1_ORYSI; Q0E4K1,CATA1_ORYSJ; Q01297, CATA1_RICCO; P30264, CATA1_SOLLC; P49284,CATA1_SOLTU; P29756, CATA1_SOYBN; P49319, CATA1_TOBAC; Q43206,CATA1_WHEAT; P25819, CATA2_ARATH; 061235, CATA2_CAEEL; P48351,CATA2_CUCPE; P30567, CATA2_GOSHI; P55308, CATA2_HORVU; P12365,CATA2_MAIZE; P49316, CATA2_NICPL; A2YH64, CATA2_ORYSI; Q0D9C4,CATA2_ORYSJ; P49318, CATA2_RICCO; Q9XHH3, CATA2_SOLLC; P55312,CATA2_SOLTU; P55313, CATA2_WHEAT; Q42547, CATA3_ARATH; P48352,CATA3_CUCPE; P18123, CATA3_MAIZE; P49317, CATA3_NICPL; O48560,CATA3_SOYBN; O48561, CATA4_SOYBN; O28050, CATA_ARCFU; P90682,CATA_ASCSU; P78574, CATA_ASPFU; Q9AXH0, CATA_AVIMR; P45737, CATA_BACFR;P14412, CATA_BACST; P26901, CATA_BACSU; P0A324, CATA_BORBR; P0A325,CATA_BORPA; P0A323, CATA_BORPE; P55304, CATA_BOTCI; P00432, CATA_BOVIN;P0A327, CATA_BRUAB; P0A326, CATA_BRUME; Q8FWU0, CATA_BRUSU; Q2I6W4,CATA_CALJA; Q59296, CATA_CAMJE; O13289, CATA_CANAL; Q96VB8, CATA_CANBO;O97492, CATA_CANFA; P07820, CATA_CANTR; Q9M5L6, CATA_CAPAN; O31066,CATA_CAUCR; Q64405, CATA_CAVPO; Q9PT92, CATA_DANRE; Q59337, CATA_DEIRA;Q9ZN99, CATA_DESVM; O77229, CATA_DICDI; P17336, CATA_DROME; P13029,CATA_ECOLI; P55305, CATA_EMENI; Q8X1P0, CATA_ERYGR; P44390, CATA_HAEIN;059651, CATA_HALMA; 073955, CATA_HALSA; P45739, CATA_HELAN; Q9ZKX5,CATA_HELPJ; P77872, CATA_HELPY; P04040, CATA_HUMAN; P07145, CATA_IPOBA;P30265, CATA_LACSK; Q9WXB9, CATA_LEGPN; Q926X0, CATA_LISIN; Q8Y3P9,CATA_LISMO; P24168, CATA_LISSE; 093662, CATA_METBF; P29422, CATA_MICLU;P24270, CATA_MOUSE; P46817, CATA_MYCBO; O08404, CATA_MYCFO; Q04657,CATA_MYCIT; A0R609, CATA_MYCS2; P00580, CATA_MYCSM; Q08129, CATA_MYCTU;Q59602, CATA_NEIGO; Q27710, CATA_ONCVE; P25890, CATA_PEA; P11934,CATA_PENJA; P32290, CATA_PHAAU; P30263, CATA_PICAN; 062839, CATA_PIG;Q5RF10, CATA_PONPY; P42321, CATA_PROMI; O52762, CATA_PSEAE; Q59714,CATA_PSEPU; Q9PWF7, CATA_RANRU; PO4762, CATA_RAT; P95631, CATA_RHIME;P37743, CATA_RHOCA; Q8Z303, CATA_SALTI; P17750, CATA_SALTY; P55306,CATA_SCHPO; P55310, CATA_SECCE; O24339, CATA_SOLAP; P55311, CATA_SOLME;Q2FH99, CATA_STAA3; Q2FYU7, CATA_STAA8; Q2YXT2, CATA_STAAB; Q5HG86,CATA_STAAC; Q99UE2, CATA STAAM; Q7A5T2, CATA_STAAN; Q6GH72, CATA_STAAR;Q6G9M4, CATA_STAAS; Q9L4S1, CATA_STAAU; Q8NWV5, CATA_STAAW; Q2PUJ9,CATA_STAEP; Q5HPK8, CATA_STAEQ; Q8CPD0, CATA_STAES; Q4L643, CATA_STAHJ;Q49XC1, CATA_STAS1; Q9KW19, CATA_STAWA; Q9EV50, CATA_STAXY; Q9Z598,CATA_STRCO; Q9XZD5, CATA_TOXGO; Q9KRQ1, CATA_VIBCH; O68146, CATA_VIBF1;Q87JE8, CATA_VIBPA; Q8D452, CATA_VIBVU; Q7MFM6, CATA_VIBVY; P15202,CATA_YEAST; Q9X6B0, CATA_YERPE; Q9Y7C2, CATB_AJECA; Q92405, CATB_ASPFU;Q877A8, CATB_ASPOR; P78619, CATB_EMENI; Q59635, CATB_PSEAE; P46206,CATB_PSESY; Q66V81, CATB_STAXY; Q9RJH9, CATB_STRCO; O87864, CATB_STRRE;P30266, CATE_BACPF; P42234, CATE_BACSU; P21179, CATE_ECOLI; P50979,CATE_MYCAV; Q9I1W8, CATE_PSEAE; P95539, CATE_PSEPU; Q9X576, CATE_RHIME;P55303, CATR_ASPNG; P06115, CATT_YEAST; P94377, CATX_BACSU; P80878,MCAT_BACSU; Q97FE0, MCAT_CLOAB; P60355, MCAT_LACPL; Q9LRS0, GOX1_ARATH;Q9LRR9, GOX2_ARATH; P05414, GOX_SPIOL; Q9UJM8, HAOX1_HUMAN; Q9WU19,HAOX1_MOUSE; Q3ZBW2, HAOX2_BOVIN; Q9NYQ3, HAOX2_HUMAN; Q9NYQ2,HAOX2_MOUSE; Q07523, HAOX2_RAT, the disclosures of which areincorporated by reference herein.

In one embodiment, a spray-dried preparation of Pichia suitable forbiocatalysis is provided. In another embodiment, a spray-driedpreparation of Saccharomyces suitable for biocatalysis is provided.Nevertheless, as yeast have cell walls, it is envisioned thatspray-dried preparations of yeast other than Pichia or Saccharomyces maybe employed for biocatalysis. In one embodiment, the yeast comprises atleast one recombinant enzyme. For example, the recombinant enzyme may bea heterologous glycolate oxidase or a heterologous catalase. In oneembodiment, the yeast comprises a heterologous glycolate oxidase and aheterologous catalase. In one embodiment, the yeast does not express arecombinant enzyme, e.g., wild-type (or otherwise nonrecombinant) yeastsuch as Saccharomyces may be employed for biocatalysis.

In one embodiment, a spray-dried preparation of prokaryotic cells suchas E. coli for biocatalysis is provided. For example, a recombinant E.coli strain comprises at least one recombinant enzyme. In oneembodiment, the E. coli strain is transformed with a prokaryotic vectorderived from pET32 into which a plant glycolate oxidase open readingframe is inserted.

To prepare recombinant strains of microbes, the microbial genome isaugmented or a portion of the genome is replaced with an expressioncassette. For biocatalysis, the expression cassette comprises a promoteroperably linked to an open reading frame for at least one enzyme thatmediates the biocatalysis. For example, the expression cassette mayencode a heterologous glycolate oxidase. In one embodiment, themicrobial genome is transformed with at least two expression cassettes,e.g., one expression cassette encodes a heterologous glycolate oxidaseand another encodes a heterologous catalase. The expression cassettesmay be introduced on the same or separate plasmids or the same ordifferent vectors for stable integrative transformation.

Recombinant or native (nonrecombinant) microbes expressing one or moreenzymes that mediate a particular enzymatic reaction are expanded toprovide a microbial cell suspension. In one embodiment, the suspensionmay be separated into a liquid fraction and a solid fraction whichcontains the cells, e.g., by centrifugation or use of a membrane, priorto spray drying. The microbial cell suspension is spray-dried underconditions effective to yield a spray-dried microbial cell preparationsuitable for biocatalysis. In one embodiment, the spray drying includesheating an amount of the cell suspension flowing through an aperature.The conditions described in the examples below were set based onsmall-scale instrument capacity, e.g., low evaporation capacity (e.g.,1.5 Kg water/hour). Thus, the ranges below are exemplary only and may bedifferent at a manufacturing scale where evaporation capacity may reachover 1000 Kg water/hour. For example, for a small scale instrument, thefeed (flow) rate may be about 1 mL/minute to about 30 mL/minute, e.g.,about 2 mL/minute up to about 20 mL/minute. Flow rates for large scaleprocesses may be up to or greater than 1 L/minute. In one embodiment,the suspension is dried at about 50° C. up to about 225° C., e.g., about100° C. up to about 200° C. The cell suspension prior to spray dryingmay be at about 5 mg/L up to about 800 mg/L, for instance, about 20 mg/Lup to about 700 mg/L, or up to or greater than 2000 mg/L. The upperlimit of the concentration of cells employed is determined by theviscosity of the cells and the instrument. The microbial cell suspensionmay be an E. coli cell suspension, a Pseudomonas cell suspension, aPichia cell suspension or a Saccharomyces cell suspension. In oneembodiment, air flow is about 500 L/hr to about 800 L/hr, e.g., about700 L/hr. The process to prepare a spray-dried microbial cellpreparation may include any flow rate, any temperature and any cellconcentration described herein, as well as other flow rates,temperatures and cell concentrations.

Once desirable native or recombinant microbial cells are spray-dried,they may be stored for any period of time under conditions that do notsubstantially impact the activity or cellular location of enzymes to beemployed in biocatalysis. Storage periods include hours, days, weeks,and up to at least 2 months.

In one embodiment, the yeast cells to be spray dried and used inbiocatalysis are Pichia cells having spinach glycolate oxidase DNAexpressed from an Aox1 promoter (single recombinant), or Pichia cellshaving spinach glycolate oxidase DNA and Saccharomyces catalase T DNAexpressed from Aox1 and Aox2 promoters (double recombinant), whichrecombinant cells are robust unicellular methylotrophic yeast. Thosecells exhibit prokaryotic growth rates, produce the heterologous enzymesintracellularly, have high production rates (grams per liter), havecontrollable expression (induced by MeOH/repressed by glycerol), andtheir fermentation is readily scaled up to 100 L. The use of those cellsis described below.

The invention will be further described by the following non-limitingexamples.

Example 1 Spray-Drying

Equipment

A Buchi B-190 spray-dryer with a pneumatic nozzle cleaner was used forall spray-drying procedures. Controlled operating parameters included:feed concentration, feed rate, air flow, temperature, and vacuum. Forall experiments, the air flow was set to 700 L/hour and the vacuum wasconstant at −50 mbar. Experimental feed concentrations ranged from 30 to600 mg/mL, feed rates spanned from 5 to 15 mL/minute, and operatingtemperatures were tested from 120 to 195° C.

Procedure

Pichia pastoris cells (designated single or double recombinant) werewashed, centrifuged, and stored at −80° C. These cells were obtainedfrom the freezer, allowed to thaw at room temperature, and excess waterwas removed by blotting the cells on filter paper. The blotted cellswere weighed into several containers to yield desired cellconcentrations after dilution to a fixed volume. The blotted cells werediluted to 100 mL total volume with deionized water. The containers wereagitated, forming cell suspensions at specified concentrations to beused as feeds to the spray-dryer.

Air flow was initiated to the spray-dryer at 700 L/hour, the aspiratorwas set to −50 mbar, and the heater was powered on. Deionized water waspumped through the spray nozzle in place of the cell suspension. Thefeed rate was set to the desired experimental value, and the heater wasadjusted to yield the correct operating temperature. After reachingtemperature equilibrium, the deionized water feed was replaced by thespecified cell suspension. The spray-drying process lasted between 6 and20 minutes based on the feed rate used. When the 100 mL cell suspensionhad been processed, the feed pump and heater were stopped, and air flowwas allowed to cool the machine to 70° C. Then, the aspirator and airsupply were shut down. The collection vessel was removed, and the Pichiacells were transferred to a scintillation vial. These vials were storedat ambient temperature in a desiccator. The feed line was flushed withdeionized water and the glassware rinsed before spray-drying at otherconditions.

Enzyme Assays

Glycolate Oxidase Assay

-   -   1. Weigh approximately 40 mg spray-dried cells (record exact        weight) into a 10 mL centrifuge tube.    -   2. Add 8.0 mL of DCIP assay solution (0.12 mM        2,6-dichloroindophenol and 80 mM Tris, pH 8.3). A DCIP solution        that is stored at 4° C. may be used for up to 2 weeks.    -   3. Mix to suspend cells, then remove 50 μL of suspension (about        0.25 mg dry cells) and place in 3.0 mL quartz cuvette with flea        stirrer. Add 2.0 mL of DCIP assay solution. Cap with septum and        bubble with nitrogen for 3 minutes.    -   4. Add 40 μL of 1.0 M L-lactic acid/1.0 M Tris (pH 8.3) to        cuvette by syringe and measure change in absorbance at 606 nm        for 30 seconds with stirring (ε=22000 L mol⁻¹ cm⁻¹).

${{Glycolate}\mspace{14mu}{oxidase}\mspace{14mu}{activity}\mspace{14mu}\left( {U\text{/}g\mspace{14mu}{dry}\mspace{14mu}{cell}} \right)} = \frac{\begin{matrix}{\left( {\Delta\;{Abs}\text{/}\min} \right)*\left( {0.00209\mspace{14mu} L} \right)*} \\\left( {1*10^{6}\mspace{14mu}{µmol}\text{/}{mol}} \right)\end{matrix}}{\begin{matrix}\begin{matrix}{\left( {22000\mspace{14mu} L\text{/}{mol}*{cm}} \right)*} \\{\left( {1\mspace{14mu}{cm}} \right)*\left( {{ca}\mspace{14mu} 0.25\mspace{14mu}{mg}} \right)*}\end{matrix} \\\left( \frac{1\mspace{14mu} g}{1000\mspace{14mu}{mg}} \right)\end{matrix}}$

Catalase Assay

-   -   1. Weigh approximately 40 mg spray-dried cells (record exact        weight) into a 10 mL centrifuge tube.    -   2. Add 8.0 mL of catalase assay buffer (0.0167 M phosphate        buffer, pH 7).    -   3. Mix to suspend cells, then, remove 50 μL of suspension (about        0.25 mg dry cells) and place in a 3.0 mL quartz cuvette with        flea stirrer. Add 2.0 mL of catalase assay buffer.    -   4. Add 1.0 mL of hydrogen peroxide solution (67 μL of 30%        peroxide in 10 mL assay buffer, pH 7.0) and measure change in        absorbance at 240 nm for 30 seconds with, stirring (ε=39.4 L        mol⁻¹ cm⁻¹).

${{Catalase}\mspace{14mu}{activity}\mspace{14mu}\left( {U\text{/}g\mspace{14mu}{dry}\mspace{14mu}{cells}} \right)} = \frac{\begin{matrix}{\left( {{\Delta Ab}\text{/}\min} \right)*\left( {0.00305\mspace{14mu} L} \right)*} \\\left( {1*10^{6}\mspace{14mu}{µmol}\text{/}{mol}} \right)\end{matrix}}{\begin{matrix}{\left( {39.4\mspace{14mu} L\text{/}{mol}*{cm}} \right)*\left( {1\mspace{14mu}{cm}} \right)*} \\{\left( {{ca}\mspace{14mu} 0.50\mspace{14mu}{mg}} \right)*\left( \frac{1\mspace{14mu} g}{1000\mspace{14mu}{mg}} \right)}\end{matrix}}$Experiment #1

This experiment investigated the feasibility of spray-drying transformedPichia cells for glycolate oxidase (GO) and catalase activity, as thetemperature shifts associated with the spray-drying process maybreak-open the Pichia cell membranes and rapid moisture loss maystabilize the recombinant enzymes. Single recombinant Pichia pastoris(NRRL Y-21001) expressing recombinant GO and native catalase was used.The study was designed to screen feed concentration, feed rate, andoperating temperature for their relative effects on GO and catalaseactivity.

A full-factorial experiment was designed with three factors (feedconcentration, feed rate, and operating temperature). A high and lowvalue was investigated for each of the three factors. This resulted in atotal of eight experimental runs shown in Table 1.

TABLE 1 Temperature Feed Rate Feed Concentration Run Pattern [C.][mL/min] [mg/mL] 601 −−− 120 5 30 602 −−+ 120 5 100 603 −+− 120 15 30604 −++ 120 15 100 605 +−− 150 5 30 606 +−+ 150 5 100 607 ++− 150 15 30608 +++ 150 15 100GO and catalase enzyme activities were the two responses used to screenthese spray-dryer operating parameters. After the spray-dried sample wasprepared for enzyme assay, three absorbance measurements were taken.Software

JMP statistical software (SAS Company) was used to plan the screeningexperiment and analyze data. The constructed model incorporated feedconcentration, feed rate, temperature, and all three interactionparameters (feed concentration*feed rate, feedconcentration*temperature, and feed rate*temperature). Standard leastsquares analysis was used to minimize residual error. Three absorbancemeasurements for each experimental condition (two replicates) wereincluded in the statistical analysis. Leverage plots are shown to gaugerelative influence of the spray-drying parameters on GO and catalaseenzyme activity.

Results

All three spray-dryer operating parameters were significant for GOactivity shown by the 95% confidence intervals crossing the horizontalaxes. Increasing the temperature from 120 to 150° C. had a slightnegative effect on GO activity, while increasing the feed rate from 5 to15 mL/minute had the steepest leverage slope and the most significanteffect on GO activity (FIGS. 3-4). Increasing the feed concentrationfrom 30 to 100 mg/mL also raised GO activity (FIG. 5). Thetemperature*feed rate interaction plot (not shown) suggested thatincreasing the feed rate would minimize GO activity loss incurred whenspray-drying at higher temperatures.

The spray-dryer operating parameters were also significant for catalaseactivity. Increasing the temperature from 120 to 150° C. had a moredrastic decrease on catalase activity compared to GO activity (FIG. 6).Consistent with GO results, increasing the feed rate from 5 to 15mL/minute had the greatest impact on catalase activity (FIG. 7).Elevating the feed concentration from 30 to 100 mg/mL raised catalaseactivity (FIG. 8). The temperature*feed rate and temperature*feedconcentration interaction plots (not shown) suggested that increasingthe feed rate and feed concentration would minimize the catalaseactivity loss from spray-drying at higher temperatures.

Experiment #2

After demonstrating feasibility of spray-drying transformed Pichia cellsfor biocatalysis, a second experiment focused on increasing GO andcatalase enzyme activity. When considering results from experiment #1for both enzymes: a) increasing temperature showed a decrease inactivity, b) elevating feed rate had the greatest positive effect onactivity, and c) increasing feed concentration raised activity. Based onthese results, the feed rate was set to 15 mL/minute (instrumentmaximum) for the second experiment. Lower temperatures were notinvestigated because spray-drying at 120° C. produced a “caked” powder(due to residual moisture) when operating at higher feed rates. As analternative, higher feed concentrations were studied to retain enzymeactivity when spray-drying above 120° C. Double recombinant Pichiapastoris expressing GO, native catalase, and catalase T fromSaccharomyces cerevisiae was used.

A full factorial experiment was designed with two factors (temperatureand feed concentration). Three different temperatures and threedifferent feed concentrations were tested. This resulted in a total ofnine experimental runs shown in Table 2.

TABLE 2 Temperature Feed Concentration Run Pattern [C.] [mg/mL] 702 11120 100 703 12 120 200 704 13 120 400 705 21 150 100 706 22 150 200 70723 150 400 708 31 195 100 709 32 195 200 710 33 195 400Again, GO and catalase enzyme activities were the two responses used toscreen these spray-dryer operating parameters. After the spray-driedsample was prepared for enzyme assay, six absorbance measurements werecompleted.

JMP statistical software was used to develop a model based ontemperature, feed concentration, and one interaction parameter(temperature*feed concentration). Run 702 (120° C., 100 mg/mL) and run705 (150° C., 100 mg/mL) were operating parameters repeated from thefirst experiment. Higher feed concentrations and higher operatingtemperatures were tested. The maximum controlled temperature (at thespecified feed rate) was 195° C. This upper temperature limit wasstudied to enhance results from increasing the feed concentration and todetermine a temperature optimum. Six absorbance measurements for eachexperimental condition (5 replicates) were included in the statisticalanalysis. Leverage plots are shown for GO and catalase enzyme activityvs. the spray-drying parameters.

Results

For the second experiment, spray-drying at 195° C. resulted insignificantly lower GO activity compared to the 120 and 150° C.conditions (FIG. 9). The benefit of spray-drying at higher feedconcentrations is not evident by observing the feed concentrationleverage plot (FIG. 10). The 95% confidence interval does not cross thehorizontal axis, and increasing the feed concentration does not show astatistically significant change in GO activity. However, there are twobenefits to increasing the feed concentration: a) more P. pastoris cellscan be processed per unit volume, and b) the temperature*feedconcentration interaction plot shows higher feed concentrations helpedachieve comparable GO activity for the 120 and 150° C. runs (FIG. 11).These results are important because spray-drying at 150° C. yields amore uniform biocatalyst powder with less residual moisture thanoperating at 120° C.

Following GO results, spray-drying at 195° C. significantly decreasedcatalase activity compared to lower operating temperatures (FIG. 12).Spray-drying at 120 and 150° C. yielded comparable catalase activity.Increasing the feed concentration provided no statistical change incatalase activity, demonstrated by the feed concentration plot and thetemperature*feed concentration interaction plot (FIGS. 13-14). Thecatalase enzyme did not receive the same benefit as GO when spray-driedat higher feed concentrations. Catalase could be more resilient totemperature increases from 120 to 150° C., and therefore not need higherfeed concentrations to maintain enzyme activity. Spray-drying P.pastoris cells up to 400 mg cells/mL (wet cell weight) can beaccomplished without loss in GO or catalase activity.

Experiment #3

The first experiment demonstrated that increasing feed rate retained GOand catalase enzyme activity in spray-dried cells. The feed rate was setto the instrument maximum (15 mL/minute) for experiment #2 where highertemperatures and feed concentrations were tested. Temperatures of 120and 150° C. yielded similar enzyme activities, but the 150° C. conditiongave a more uniform and dry biocatalyst powder. The maximum operatingtemperature of 195° C. was studied, and clearly, there exists an uppertemperature limit for best enzyme activity. An upper limit on feedconcentration could not be determined from the second experiment wherefeeds up to 400 mg/mL were tested without any statistical change inenzyme activity.

For the third experiment, the best feed rate (15 mL/minute) and besttemperature (150° C.) from previous studies were used. Increasing feedconcentration can be beneficial by reducing processing volume andhelping maintain enzyme activity if spray-drying at temperatures above150° C. was used. Feed concentrations of 200 and 400 mg/mL were repeatedand 600 mg/mL was also investigated. The viscosity of the 600 mg/mLconcentration slowed the feed rate to 13 mL/minute. Concentrations above600 mg/mL were not tested because earlier experiments proved high feedrates yielded better enzyme activities than increasing feedconcentration. Table 3 below is a summary of conditions for experiment#3.

TABLE 3 Feed Concentration Run [mg/mL 712 200 713 400 714 600Further StudiesEnzyme stability studies, cell permeabilization studies, enzyme leachingstudies, and enantiomeric specificity studies were also investigated.

Enzyme Stability.

The spray-dried cells were stored at room temperature in a desiccator(to prevent moisture contamination). These cells were assayed for enzymeactivity over a 17-day period to observe possible activity loss andquantify enzyme stability.

Cell Permeabilization.

In initial work (in the absence of spray-drying), after fermentation,cells were recovered by solid/liquid separation, and benzalkoniumchloride (BAC) detergent was used to permeabilize cells for the enzymereaction. Nevertheless, the results from experiment #1 hereinaboveshowed the feasibility of using spray-dried cells for GO and catalaseactivity. Thus, another experiment (see below) compared spray-driedcells to several other methods of cell preparation: a) not spray-driedand not BAC treated, b) not spray-dried and treated with BAC, and c)spray-dried and treated with BAC. The spray-dried cells were treatedwith BAC after spray-drying to determine if spray-drying completelypermeabilized the cells.

Enzyme Leaching.

It is beneficial to have GO and catalase contained within the whole cellbiocatalyst. The cells offer protection from shear forces in thereactor, keep the enzymes spatially close so they can work together, andfacilitate enzyme recovery for reuse. For these reasons, leaching of GOand catalase from spray-dried cells was investigated.

Methods

Detergent Cell Permeabilization

-   -   1. Prepare mixture of 10 weight % cells/volume 50 mM phosphate        buffer (pH 7.0) at room temperature. Add 0.1% (w/v) benzalkonium        chloride.    -   2. Stir or mix slowly for 60 minutes at room temperature, 25° C.    -   3. Centrifuge (9950×g for 10 minutes at 5° C.), decant, and        re-suspend cells three times (10% w/v) in 50 mM phosphate buffer        (pH 7.0) at 5° C., centrifuge, decant.    -   4. Permeabilized cells may be used immediately or stored up to        one year at −80° C. prior to use.

Glycolate Oxidase Leaching Assay

-   -   1. Weigh approximately 40 mg spray-dried cells (record exact        weight) into a 10 mL centrifuge tube.    -   2. Add 8.0 mL of DCIP assay solution (0.12 mM        2,6-dichloroindophenol and 80 mM Tris, pH 8.3).    -   3. Mix to suspend cells, then remove 50 μL of suspension (about        0.25 mg dry cells) and place in 3.0 mL quartz cuvette with flea        stirrer. Add 2.0 mL of DCIP assay solution. Cap with septum and        bubble with nitrogen for 3 minutes.        -   * When testing supernatant (for enzyme leaching), use 2.05            mL of supernatant and do not dilute further with assay            buffer.    -   4. Add 40 μL of 1.0 M glycolic acid (or L-lactic acid)/1.0 M        Tris (pH 8.3) to cuvette by syringe and measure change in        absorbance at 606 nm for 30 seconds with stirring (ε=22000 L        mol⁻¹ cm⁻¹).    -   5. Calculate the total glycolate oxidase activity.    -   6. Centrifuge cell suspension at 9950×g for 10 minutes. Measure        enzyme activity of supernatant (without buffer dilution).    -   7. Re-suspend cell pellet in 6.0 mL assay buffer and repeat        enzyme assay on cell suspension and supernatant.

Catalase Leaching Assay

-   -   1. Weigh approximately 40 mg spray-dried cells (record exact        weight) into a 10 mL centrifuge tube.    -   2. Add 8.0 mL of catalase assay buffer (0.0167 M phosphate        buffer, pH 7).    -   3. Mix to suspend cells, then remove 50 μL of suspension (about        0.25 mg dry cells) and place in a 3.0 mL quartz cuvette with        flea stirrer. Add 2.0 mL of catalase assay buffer.        -   * When testing supernatant (for enzyme leaching), use 2.05            mL of supernatant and do not dilute further with assay            buffer.    -   4. Add 1.0 mL of hydrogen peroxide solution (67 μL of 30%        peroxide in 10 mL assay buffer, pH 7.0) and measure change in        absorbance at 240 nm for 30 s with stirring (ε=39.4 L mol⁻¹        cm⁻¹).    -   5. Calculate the total catalase activity.    -   6. Centrifuge cell suspension at 9950 g for 10 minutes. Measure        enzyme activity of supernatant (without buffer dilution).    -   7. Re-suspend cell pellet in 6.0 mL assay buffer and repeat        enzyme assay on cell suspension and supernatant.        Results

Enzyme Activity

The feed rate was held constant at 15 mL/minute and temperature was setto 150° C. Conditions 712, 713, and 714 had feed concentrations of 200,400, and 600 mg cells/mL, respectively. Six absorbance measurements (5replicates) were taken for each condition and used to calculate enzymeactivities. The six enzyme activities were plotted along with anadjusted average. The adjusted average is an average of the four middleenzyme activities (dropping the highest and lowest numbers). All threeconditions tested had overlapping GO activities, but the adjustedaverages increased slightly with each successive feed concentration(FIG. 15). The catalase assay showed less variability, and the 600 mg/mLcondition clearly had the highest resulting catalase activity (FIG. 16).Feed concentrations up to 600 mg/mL were spray-dried resulting in highenzyme activities and low processing volumes. Higher feed concentrationswere not studied because their viscosities decreased feed rates belowthe 15 mL/minute optimum.

Enzyme Stability

Initial work used BAC detergent to permeabilize cells instead ofspray-drying, and BAC treated cells required storage at −80° C. tomaintain enzyme activity. Spray-dried cells were stored in a desiccatorat room temperature. The spray-dried cells were sampled for enzymeactivity three times over a 17-day period. For each sampled condition,six absorbance measurements were taken. The average value was plottedwith error bars signifying one standard deviation. Considering GO andcatalase, no statistical loss in activity was observed over the 17-daysampling period (FIGS. 17-18).

Cell Permeabilization

The current method of spray-drying cells for enzyme activity wascompared to initial work using BAC detergent permeabilization. Again,each condition had six absorbance measurements. The average was plotted,and error bars show one standard deviation. All BAC treated cells werepermeabilized and washed according to “BAC Detergent Permeabilization.”Because BAC treatment involves phosphate buffer, all permeabilized cellswere assayed using 40 mg blotted (wet) cells. Spray-dried cells that hadnot been permeabilized were assayed using 40 mg dry cell weight, and sohad more enzyme per given cell mass. While a direct comparison betweenpermeabilized cells and spray-dried cells cannot be made, two importantconclusions can be understood. Cells that had not been spray-driedrequired BAC permeabilization to reach useful levels of GO and catalaseenzyme activity, but all spray-dried cells had appreciable levels ofenzyme activity without added detergent (FIGS. 19-20). Second, cellsthat were permeabilized post spray-drying showed lower GO activity butgave comparable/higher catalase activity meaning BAC detergent could betoxic to the GO enzyme.

TABLE 4 Effect of benzalkonium chloride on the GO activity of postspray-dried (SD) pichia pastoris BAC concentration Average GO activityin IU/g spray-dried cells (w/v) Lactic acid 3-Ph-lactic acid2-OH-butyric acid SD cells* 132 34 146 0% BAC 139 39 162 0.02% BAC 14336 162 0.1% BAC 149 35 185 *These assays were done with 4 days old DCIPassay solution

Leaching

Operating conditions to study leaching were 150° C., 600 mg cells/mL,and 13 mL/minute (low feed rate due to high feed viscosity). Threeabsorbance readings were taken, resulting enzyme activities wereplotted, and the group average or statistical mean was shown. Thisprocess was repeated after three separate washings and the cellsuspension and wash supernatant were tested for enzyme activity. Plotsof enzyme activity for the cell suspension are shown in FIGS. 21-22. Nostatistical change in GO or catalase enzyme activity was observed aftereach of the three washings. Furthermore, no measurable enzyme activitywas detected in the wash supernatant (not shown). This is a strongindication that spray-dried cells will not leach GO and catalase enzymesand can be recycled through the enzyme reaction environment.

Specificity

The specificity of glycolate oxidase for various substrates andenantiomers is shown in Table 5 and FIG. 23.

TABLE 5 Comparison of relative GO activities where lactic acid is 100%Racemic 2-hydroxy- Relative activity in % carboxylic acids PermeabilizedSpray-dried Lactic acid 100 100 3-Indolelactic acid 18 26 3-Phenyllacticacid 25 37 p-Hydroxyphenyllactic acid 26 38 β-Chlorolactic acid 100 121Trifluorolactic acid 11 2 2-Hydroxybutyric acid 91 1292-Hydroxy-3-methylbutyric acid 1 2 Glycolic acid 90 94 Mandelic acid 3 32-Hydroxydecanoic acid 40 49

In summary, spray drying of Pichia cells renders it porous forbiocatalysis, without the need for any enzyme purification orprocessing. Moreover, maximum activity is achieved with many substrateswithout treatment with porosity agents, the spray-dried cells andenzymes are stable for up to 30 days at room temperature. Further, thereaction is fully enantioselective with respect to glycolate oxidase.

Example 2 Expression of GO in E. Coli

The GO gene was cloned into the E. coli expression plasmid pET-32a(Novagen). A forward PCR primer (GO-AseI-F,5′GGCTCGGATTAATGGAGATCACAAATGTGAACG-3; SEQ ID NO:1) and a reverse PCRprimer (GO-XhoI-R, 5′-GCATGCCTCGAGTTATAATCTGGCAACAGCACG-3; SEQ ID NO:2)were used for PCR amplification of the GO gene from plasmid pPM1 (Payneet al., Gene, 167:215 (1995)). The 1.1 kb PCR product was first digestedwith AseI and XhoI and subsequently ligated with a 5.4 kb fragment ofpET-32a obtained from a NdeI-XhoI digestion. The resultant recombinantplasmid was named as pET-GO#11. DNA sequencing of pET-GO#11 showed thatthe cloned GO gene had a single point mutation (probably resulted fromPCR amplification) which did not alter the GO protein sequence.Therefore, pET-GO#11 was transformed into E. coli Rosetta-gami B(DE3)cells (Novagen) for protein expression.

A 5 mL seed culture of Rosetta-gami B(DE3)/pET-GO#11 cells was grown inLB medium supplemented with ampicillin (100 μg/mL) and chloramphenicol(34 μg/mL) at 37° C. for 16 hours. The seed culture was used toinoculate 250 mL LB-ampicillin-chloramphenicol medium to an initial celldensity of 0.01 OD₆₀₀ (optical density at 600 nm) units. The culture wasincubated at 30° C. with shaking at 230 rpm, until the OD₆₀₀ valuereached 0.3. Incubation was continued at 24° C. with shaking at 230 rpmuntil the OD₆₀₀ value reached 0.5 to 0.6.Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to the culture(final concentration of 0.2 mM) to induce GO protein production. Theinduced culture was incubated at 24° C. with shaking at 230 rpm for 16hours. The cells were harvested by centrifugation at 10,000×g, 4° C. for10 minutes. The cell pellet was then suspended in 10 mL phosphatebuffered saline (PBS) plus 1 mM dithiothreitol and 1 mMphenylmethylsulfonyl fluoride. The cells were disrupted by passingthrough a chilled French pressure cell two times at 260 MPa. The lysatewas centrifuged at 25,800×g 4° C. for 10 minutes, and supernatant thatcontained 3.7 mg of protein/mL (determined by Bradford assay with BSA asstandard) was saved as a cell extract for a GO activity assay (FIG. 25)and SDS-PAGE analysis (FIG. 24).

GO activity of a Rosetta-gami B(DE3)/pET-GO#11 cell extract was measuredas described above. GO activity of a Rosetta-gami B(DE3)/pET-GO#11 cellextract was tested (a) with substrate (lactate), (b) without substrate,and (c) with substrate and a thermally denatured extract (by boilingextract at 110° C. for 10 minutes). These results were compared to acontrol extract from Rosetta-gami B(DE3) cells without the pET-GO#11plasmid under the same assay conditions. Results were plotted in enzymeactivity units (U)/mg protein.

The Rosetta-gami B(DE3)/pET-GO#11 soluble fraction had sufficient GOactivity to generate more cell mass for spray-drying. A 100 mL seedculture of Rosetta-gami B(DE3)/pET-GO#11 cells were grown in LB mediumsupplemented with ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL)at 37° C. for 16 hours. The seed culture was used to inoculate 500 mLLB-ampicillin-chloramphenicol medium to an initial cell density of 0.1OD₆₀₀ (optical density at 600 nm) units. The culture was incubated at29° C. with shaking at 180 rpm, until the OD₆₀₀ value reached 0.3.Incubation was continued at 25° C. with shaking at 230 rpm until theOD₆₀₀ value reached 0.5 to 0.6. Isopropyl-β-D-thiogalactopyranoside(IPTG) was then added to the culture (final concentration of 0.2 mM) toinduce GO protein production. The induced culture was incubated at 25°C. with shaking at 230 rpm for 16 hours. The cells were harvested bycentrifugation at 10,000×g, 4° C. for 10 minutes. The cell pellet wasstored at −80° C. while the above growth procedure was repeated togenerate a total of 11 g wet cell weight for spray-drying studies.

The Rosetta-gami B(DE3)/pET-GO#11 cells were thawed at 4° C. The 11 gcell pellet was divided into two parts: 10 g were spray-dried, and theremaining 1 g was kept as a control. The 10 g of cells were diluted withdeionized water to a total volume of 40 mL. This yielded a cellconcentration of 250 mg/mL for the spray-dryer feed. Spray-dryeroperating conditions were 15 mL/minute feed rate, 600 L/hour air flowrate, and 150° C. inlet air temperature. The resulting biocatalystpowder was collected for GO activity analysis. The remaining 1 gRosetta-gami B(DE3)/pET-GO#11 cells (not spray-dried) were blotted onWhatman #2 filter paper to remove excess moisture. The spray-dried cellsand blotted (control) cells were assayed for GO activity as describedhereinabove. Results for spray-dried cells and blotted cells are shownin U/mg dry cell weight and U/mg blotted cell weight, respectively.Three cell masses were tested for each cell preparation method and GOactivity was calculated on a per gram basis. The lowest cell mass (0.25mg) was near the sensitivity limits of the spectrophotometer, resultingin higher error for the initial data point. The spray-dried Rosetta-gamiB(DE3)/pET-GO#11 cells retained nearly 10 activity units per gram drycells (FIG. 26). With additional optimization of spray-dryingparameters, i.e., operating temperature, feed rate, and feed cellconcentration, further GO activity could be achieved in spray-driedRosetta-gami B(DE3)/pET-GO#11 cells.

Another organism, Saccharomyces cerevisiae, containing native catalaseenzyme, was spray-dried and tested for catalase activity. S. cerevisiaewas obtained from SAF-Instant Company as a granular free-flowing yeastfor use in baking doughs with short fermentation processes. 10 g ofgranular S. cerevisiae cells were diluted with deionized water to atotal volume of 100 mL. This yielded a cell concentration of 100 mg/mLfor the spray-dryer feed. Spray-dryer operating conditions were 15mL/minute feed rate, 600 L/hour air flow rate, and 150° C. inlet airtemperature. The resulting biocatalyst powder was collected for catalaseactivity analysis. Another 40 mg of granular S. cerevisiae (notspray-dried) was used as a control. The spray-dried cells and controlcells were assayed for catalase activity as described hereinabove;Results for spray-dried cells and control cells were plotted as U/mg dryweight. Three cell masses were tested for each cell preparation methodand catalase activity was calculated on a per gram basis. The lowestcell mass (0.25 mg) was near the sensitivity limits of thespectrophotometer, resulting in higher error for the initial data point.The spray-dried SAF-Instant granular S. cerevisiae cells retained 25 to30 thousand activity units per gram dry cells. The S. cerevisiae thatwas not spray-dried may have been subjected to processing conditions bythe manufacturer to create the granular yeast, and those conditionscould have partially permeabilized the cell membranes and increasedcatalase activity in the non spray-dried samples.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

What is claimed is:
 1. A method to prepare a whole recombinant yeastcell biocatalyst preparation, comprising: a) providing a wholerecombinant yeast cell suspension which is up to 400 mg/mL; and b) spraydrying the recombinant yeast cell suspension in the absence of apermeabilization agent at a temperature up to 150° C. effective to yielda spray-dried recombinant yeast cell preparation suitable forbiocatalysis, wherein the spray-dried cells are porous but retain anenzyme active in the biocatalysis.
 2. The method of claim 1 wherein thespray drying includes heating an amount of the cell suspension flowingthrough an aperature.
 3. The method of claim 1 wherein the yeast cellsuspension is a Pichia, Hansenula or Saccharomyces cell suspension. 4.The method of claim 1 wherein the recombinant yeast cell comprises anexpression cassette encoding a heterologous glycolate oxidase.
 5. Themethod of claim 4 wherein the recombinant yeast cell comprises anexpression cassette encoding a heterologous catalase.
 6. The method ofclaim 1 further comprising separating the suspension into a liquidfraction and a solid fraction which contains the cells prior to spraydrying.
 7. The method of claim 6 further comprising spray drying theseparated cells.
 8. The method of claim 6 wherein the suspension isseparated by centrifugation.
 9. The method of claim 6 wherein thesuspension is separated using a membrane.
 10. A spray-dried recombinantyeast cell preparation prepared by the method of claim
 1. 11. Thepreparation of claim 10 wherein the yeast is Pichia.
 12. The preparationof claim 10 wherein the recombinant yeast comprises at least onerecombinant enzyme.
 13. The preparation of claim 12 wherein therecombinant enzyme is a heterologous glycolate oxidase.
 14. Thepreparation of claim 12 wherein the recombinant enzyme is a heterologouscatalase.
 15. The preparation of claim 10 wherein the recombinant yeasthas a genome that is augmented or a portion of the genome that isreplaced with an expression cassette.
 16. A method to prepare productsof an enzymatic reaction, comprising: a) providing the spray-driedpreparation of claim 10; and b) combining the preparation and an aqueoussolution comprising a substrate for an enzyme in the preparation underconditions that yield a product of the enzyme catalyzed reaction. 17.The method of claim 16 wherein the enzyme is glycolate oxidase.
 18. Themethod of claim 17 wherein the product is pyruvate or glyoxylate. 19.The method of claim 16 wherein the products are keto acids or chiralhydroxyl acids.
 20. The method of claim 16 further comprising isolatingthe product.